title: Regulation of Na,K-ATPase β1-subunit in TGF-β2-mediated epithelial-to-mesenchymal transition in human retinal pigmented epithelial cells.
abstract: Proliferative vitreo retinopathy (PVR) is associated with extracellular matrix membrane (ECM) formation on the neural retina and disruption of the multilayered retinal architecture leading to distorted vision and blindness. During disease progression in PVR, retinal pigmented epithelial cells (RPE) lose cell-cell adhesion, undergo epithelial-to-mesenchymal transition (EMT), and deposit ECM leading to tissue fibrosis. The EMT process is mediated via exposure to vitreous cytokines and growth factors such as TGF-β2. Previous studies have shown that Na,K-ATPase is required for maintaining a normal polarized epithelial phenotype and that decreased Na,K-ATPase function and subunit levels are associated with TGF-β1-mediated EMT in kidney cells. In contrast to the basolateral localization of Na,K-ATPase in most epithelia, including kidney, Na,K-ATPase is found on the apical membrane in RPE cells. We now show that EMT is also associated with altered Na,K-ATPase expression in RPE cells. TGF-β2 treatment of ARPE-19 cells resulted in a time-dependent decrease in Na,K-ATPase β1 mRNA and protein levels while Na,K-ATPase α1 levels, Na,K-ATPase activity, and intracellular sodium levels remained largely unchanged. In TGF-β2-treated cells reduced Na,K-ATPase β1 mRNA inversely correlated with HIF-1α levels and analysis of the Na,K-ATPase β1 promoter revealed a putative hypoxia response element (HRE). HIF-1α bound to the Na,K-ATPase β1 promoter and inhibiting the activity of HIF-1α blocked the TGF-β2 mediated Na,K-ATPase β1 decrease suggesting that HIF-1α plays a potential role in Na,K-ATPase β1 regulation during EMT in RPE cells. Furthermore, knockdown of Na,K-ATPase β1 in ARPE-19 cells was associated with a change in cell morphology from epithelial to mesenchymal and induction of EMT markers such as α-smooth muscle actin and fibronectin, suggesting that loss of Na,K-ATPase β1 is a potential contributor to TGF-β2-mediated EMT in RPE cells.
authors.affiliation: Nemours Biomedical Research, Alfred I. duPont Hospital for Children, Rockland Center I, 1701 Rockland Road, Wilmington, DE 19803, USA. Electronic address: sridevi_mony@lycos.com.
authors.forename: Sridevi
authors.initials: S
authors.lastname: Mony
authors.forename: Seung Joon
authors.initials: SJ
authors.lastname: Lee
authors.forename: Jeffrey F
authors.initials: JF
authors.lastname: Harper
authors.forename: Sonali P
authors.initials: SP
authors.lastname: Barwe
authors.forename: Sigrid A
authors.initials: SA
authors.lastname: Langhans
mesh_terms: D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects* / Q000502:physiology; D004848:Epithelium / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D015151:Immunoblotting; D018613:Microscopy, Confocal; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000201:enzymology*; D012172:Retinaldehyde / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*
chemical_list: C503551:ATP1B1 protein, human; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D000254:Sodium-Potassium-Exchanging ATPase; D012172:Retinaldehyde
keywords: HIF-1α; Na,K-ATPase beta subunit; TGF-β(2); epithelial mesenchymal transition (EMT); proliferative vitreoretinopathy (PVR); retinal pigmented epithelium (RPE)